The immune system has evolved specific mechanisms to generate diverse antibody specificities from limited amounts of genetic material. Antibody genes are generated by the somatic recombination of a small number of gene segments. Somatically recombined antibodies have low affinities for many antigens. Fine-tuning of the affinity of a particular antibody to a particular antigen is driven by somatic hypermutation, a B cell specific reaction which introduces point mutations into the variable (V) regions of immunoglobulin (Ig) genes. Currently, our mechanistic understanding of hypermutation is very limited. We, and others, have obtained results implicating DNA double strand breaks (DSBs) as reaction intermediates. We do not know how the DSBs arise, but their generation is independent of the recently discovered activation-induced cytidine deaminase. Our working hypothesis is that a B cell specific nuclease is targeted to the V regions of Ig genes creating a lesion in the DNA which is, or resolves into, a DSB. The long range goal of the proposed research is to elucidate the molecular mechanism of the hypermutation reaction. The first part of the proposed project will be to establish whether site-specific breaks of known structure can hypermutate. For this purpose, we will create lines of mice with V regions altered to contain the recognition sequence for the rare endonuclease I-Scel (a DSB nuclease) and for the nicking enzyme N.BstNB1. We will assess mutability after crossing these with lines that inducibly express either enzyme. Hypermutation (and the associated DSBs) absolutely depend on the Ig enhancers. In the second part, we will use biochemical methods to define the precise sequence elements within the enhancers which control hypermutation. We will then verify that they are functionally important by comparing Ig kappa transgenes which either retain or lack such sequences. Finally, we will define the factors which bind these elements and target the hypermutation machinery specifically to Ig genes. As aberrant targeting of hypermutation has been linked to a variety of B cell lymphomas, these studies have significant implications for understanding the generation of such malignancies.